Compounds for reversing drug resistance

ABSTRACT

The invention features novel peptide derivatives called Reversins, and provides for their use in a method of reducing the activity of the multi-drug transporter protein MDR1 in order to overcome multidrug resistance in a mammal. The peptide derivatives are of the formula (I) X 1   n -X 2 -X 3 (X 4 ) n -X 5 , wherein n is 0 or 1, and each n is the same or different; X 1  is BOC, BOC-Asu, Z-Asu, benzyloxycarbonyl, Glu(OBzl)-OBzl, Trp-OMe, Trp-Phe-OMe, Phe-Trp-OMe, Phe-Phe-OtBu, Trp-Trp-OtBu, indoloacetyl, benzoyl, an alkylanine of 1-4 carbons, dibenzylamide, tryptamide, 1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylamide or dicyclohexylamide; X 2  is Glu(OBzl), Asp(OBzl), succinyl, O,O-dibenzoyltartaroyl, diphenoyl, muconyl, Thx, Cpa, Asu, Nal, Pen, Phg, Dbt, Lys(BOC), Lys(Z), Cys(Bzl), Thr(Bzl), Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Phe, Tyr(Bzl), or Ser(Bal); X 3  is Asp, Asu, Lys, Glu, Trp, Thx, Cpa, Nal, Pen, Phg, Dbt, Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Tyr, Phe, or Tyr(Bzl); X 4  is BOC-Glu(OBzl), Glu(OBzl), Asu, OBzl, Bzl, BOC, BOC-Lys(BOC), Z-Glu(OtBu), Asp(OBzl), Asp(OBz)-OBzl, benzyloxycarbonyl, O-(cyclo-hexyl), fluorenylmethyl ester, Glu(OtBu), Glu(OtBu)-OBzl, 1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylarnide, or dicyclohexylamide; and X 5  is OMe, OBzl, OtBu, Phe-OMe, -O-(cyclohexyl), Trp-OMe, (chlorophenyl)-isobutylamide, fluorenylmethyl ester, ONp, 1-aminoadamantane, aminomethylcyclohexane, indoline, phenylethylamide, or dicyclohexylamide.

BACKGROUND OF THE INVENTION

This invention relates to compounds for overcoming resistance that apatient may build to therapeutics.

Treatment of many diseases can be severely limited by resistance to thechosen therapeutic drug. For example, chemotherapy, while generally aneffective treatment against human cancerous diseases, is hampered when apatient becomes resistant to the chemotherapeutic. In one special formof drug resistance, called “Multidrug Resistance,” the cell becomesresistant not only to the chemotherapeutic being administered, but to awide range of structurally and functionally unrelated drugssimultaneously (see Ford et al., Pharmacological Reviews, 42:155-199,1992).

The cause of multidrug resistance is the appearance of an integralglycoprotein in the plasma membrane of the targeted cell, e.g., a tumorcell (FIG. 1). The protein functions as a multidrug transporter, and isvariously called MultiDrug-Resistance 1 protein (MDR1), P-glycoprotein(pleiotropic-glycoprotein), Pgp, or P-170. MDRl consists of 1280 aminoacid residues, and contains 12 transmembrane segments and twonucleotide-binding domains. It strongly resembles prokaryotic andeukaryotic members of the so-called ABC (ATP Binding Cassette)transporters, or traffic ATPases (see Endicott et al., Annu. Rev.Biochem. 58:137-171, 1989; Higgins, Annu. Rev. Cell. Biol. 8:67-113,1992).

MDR1 naturally functions to, and is highly expressed in tissues normallyresponsible for, extruding toxic materials and waste-products from cells(e.g., lung, kidney, and liver), and secretes hydrophobic compounds fromexocrine or endocrine glands (Gottesman et al., J. Biol. Chem.263:12163-12166, 1988; Higgins et al., supra). Consistent with itsnatural function, MDR1 catalyses an ATP-dependent extrusion of variouscytotoxic drugs from the cell, e.g., vinca alkaloids, anthracyclines,and other natural antibiotics, thereby maintaining their cellular levelat a subtoxic concentration. Thus, when expressed by tumor cells, MDR1expels cytotoxic chemotherapeutic agents, and thus allows the tumor cellto survive anticancer treatments even at high drug doses. At the sametime, “ordinary” cells, having no such extrusion mechanism, may receivea lethal drug exposure. Tumors developing from tissues normallyexpressing the MDR1 protein often show a primary drug resistance, whilein other tumors a secondary drug resistance may develop afterchemotherapy.

The phenomenon of multidrug resistance is not limited to tumor cells.MDR1 and its homologues are expressed in a wide variety of cell-types,including parasitic protozoa. Consequently, overexpression of a memberof the MDR1 family of proteins creates obstacles to a wide variety ofparasitic diseases, including malaria, African sleeping sickness, andothers (Campbell et al., Chemotherapy of Parasitic Diseases, PlenumPress:NY, 1986; Henderson et al., Mol. Cell. Biol. 12:2855-65, 1992).MDR1 is also expressed by endothelial cells of human capillary bloodvessels at the blood-brain barrier and blood-testis barrier (Ford et al.supra, at 159).

It is known that verapamil, a drug that blocks voltage-dependent calciumchannels, stimulates the activity of MDR1-bound ATPase at aconcentration of 1 to 20 μM but inhibits it as a concentration above 100μM (Sarkadi et al. J. Biol. Chem. 267:4854-4858, 1992). While betweenthese concentrations verapamil blocks the extrusion of antitumor drugs,its high toxicity severely limits its clinical use (Solary et al.Leukemia 5:592-597, 1991; Dalton et al. J. Clin. Oncology 7:415-418,1989).

In SU-A-1544778, Golovina, T. N. et al describe the preparation ofdifferent peptides one of which, BOC-Leu-Tyr-OMe is structurally closeto the peptides provided by the present invention. Nevertheless, nohints on the possible biological activity of said peptide are disclosed.

SUMMARY OF THE INVENTION

The invention generally features chemical compositions which reduce orovercome multidrug resistance in a mammal, e.g., a human, and inmicroorganisms causing disease in a mammal. The compounds, called“Formula (I)” compounds, or “Reversins”, are hydrophobic peptidederivatives which effectively compete with cytostatic drugs on the MDR1protein, thus reducing or eliminating drug resistance.

“Multi-drug resistance”, as used herein, refers to the ability of cellsto develop resistance to a broad range of structurally or functionallyunrelated drugs. This occurs by outward transport of the drug from thecell, the transport being mediated by the MDR1 glycoprotein or itshomologues. The term “multidrug resistance” also applies to thecross-resistance between drugs which is adversely affected by theReversin compounds of the invention (see below). Preferably, “multidrugresistance” refers to the state which is dependent on expression oroverexpression of the MDR1 protein or its homologues, and/or on geneamplification of human mdr1 or its homologues. Both primary andsecondary multidrug resistance are included. Where the drug resistanceis “primary” the cell has experienced no previous exposure to a memberof the group of drugs, yet exhibits inherent resistance to them. Wheredrug resistance is “secondary”, the cell has been exposed to only onedrug, or to only a subset of two or more, but not necessarily to thewhole, group of drugs affected by cross-resistance.

The compounds of the invention, hereafter called “Reversins”, are offormula (I):

X¹ _(n)-X²-X³-(X⁴)_(m)-X⁵  (I)

wherein

n and m are 0 to 1;

X¹ is BOC, BOC-Asu, Z-Asu, benzyloxycarbonyl, Glu(OBzl)-OBzl, Trp-OMe,Trp-Phe-OMe, Phe-Trp-OMe, Phe-Phe-OtBu, Trp-Trp-OtBu, indoloacetyl,benzoyl, an alkylamine of 1-4 carbons, dibenzylamide, tryptamide,1-amino-adamantine, aminomethylcyclohexane, indoline, phenylethylamideor dicyclohexylamide;

X² is Glu(OBzl), Asp(OBzl), succinyl, O,O-dibenzoyltartaroyl, diphenoyl,muconyl, Thx, Cpa, Asu, Nal, Pen, Phg, Dbt, Lys(BOC), Lys(Z), Cys(Bzl),Thr(Bzl), Glu(OtBu), tert.-Leu, Leu, Nle, Pro, Phe, Tyr(Bzl), orSer(Bzl);

X³ is Asp, Asu, Lys, Glu, Trp, Thx, Cpa, Nal, Pen, Phg, Dbt, Glu(OtBu),tert.-Leu, Leu, Nle, Pro, Tyr, Phe; or Tyr(Bzl);

X⁴ is BOC-Glu(OBzl), Glu(OBzl), Asu, OBzl, Bzl, BOC, BOC-Lys(BOC),Z-Glu(OtBu), Asp(OBzl), Asp(OBz)-OBzl, benzyl-oxycarbonyl,O-(cyclo-hexyl), fluorenylmethyl ester, Glu(OtBu), Glu(OtBu)-OBzl,1-amino-adamantine, amino-methylcyclohexane, indoline, phenylethylamide,or dicyclohexylamide; and

X⁵ is OMe, OBzl, OtBu, Phe-OMe, -O-(cyclohexyl), Trp-OMe,(chlorophenyl)-isobutylamide, fluorenylmethyl ester, ONp,1-aminoadamantane, aminomethylcyclohexane, indoline, phenylethylamide,or dicyclohexylamide,

with the proviso that said formula (I) is not BOC-Leu-Tyr-OMe.

Formula (I) compounds containing amino acids with either the L or Dconfiguration fall within the scope of the invention.

Side chain protecting groups of amino acids may be substituted by one ormore halogen atoms, e.g., chloro-Z, or bromo-Z. Such blocking groups areknown. A benzyl ester group can be substituted by one or more nitrogroups in the second or fourth position of the benzene ring.

The abbreviations used herein are known to those skilled in the art(see, e.g., J. Biol. Chem. 241:527, 1966; J. Biol. Chem. 247:977 1972;hereby incorporated by reference). Other abbreviations used herein areas follows:

AM: acetoxymethyl ester Asu: aminosuccinic acid or aminosuccinoyl BOC:tert.-butyloxycarbonyl, Bzl: benzyl, Cpa: 4-chlorophenylalanyl, Cys:cysteinyl, Dbt: dibromotyrosyl, DBTA: dibenzoyltartaroyl, DCC:dicyclohexylcarbodiimide, DCU: dicyclohexylurea, DIC:diisopropylcarbodiimide, DMF: dimethylformamide HPLC: high pressureliquid chromatography, MDR1: product of multidrug resistance gene, Me:methyl, Nal: naphthylalanyl, Nle: norleucyl, m.p.: melting point, OPFP:pentailuorphenyl ON_(p): p-nitrophenyl Pen: penicillinalanyl, Phe:phenylalanyl, Phg: phenylglycyl, Pro: prolyl, R_(f): retention factor,SUC: succinyl, TEA: triethylamine, THF: tetrahydrofuran, Thr: threonyl,Thx: thyroxyl, TLC: thin layer chromatography, Trp: tryptophyl, Z:benzyloxycarbonyl.

As preferred embodiments of the invention, X¹ can be BOC,Glu(OBzl)-OBzl, Z, or (D-Phe-Trp-OMe); X² can be Asp(OBzl), succinyl,Glu(OBzl), or DBTA; X³ can be Lys, Glu, Asp, or Phe; X⁴ can be Z, OBzl,BOC-Glu(OBzl), BOC-Lys(BOC), or Z-Glu(OtBu); or X³ and X⁴ in combinationcan be Lys[BOC-Glu(OBzl)]; and X⁵ can be OtBu, OBzl, OMe, or Trp-OMe.

Preferred formula (I) compounds of the invention include, but are notlimited to, the peptide derivatives BOC-Asp(OBzl)-Lys(Z)-OtBu (Reversin121); succinylbis[Glu(OBzl)-OBzl]; Z-D-Glu(OBzl)-D-Asp(OBzl)-OBzl;DBTA-bis(D-Phe-Trp-OMe); DBTA-[Glu(OBzl)₂]₂;N^(α),N^(ε)-bis[BOC-Glu(Bzl)]-Lys-OMe (Reversin 205);BOC-Tyr(Bzl)-Tyr(Bzl)-OMe; BOC-D-Ser(Bzl)-Lys(Z)-OtBu;BOC-Glu(OBzl)-Lys(Z)-OtBu; BOC-Glu(OBzl)-Lys(Z)-OMe;N^(α),N^(ε)-bis[BOC-Lys(BOC)]-Lys-OMe; orN^(α),N^(ε)-bis[Z-Glu(OtBu)]-Lys-OMe.

Any of the various compounds of the invention can be combined with apharmaceutically acceptable carrier or an adjuvant. The variouscompounds of the invention can also be combined with a drug, e.g., achemotherapeutic, antiparasitic, or antibiotic drug, for convenientco-administration of the Reversin compound and the drug to a patient.The Reversin molecule can, in addition to inhibiting MDR1 activity, alsoact as a adjuvant to enhance the activity of the drug. As used herein, a“drug”, includes a medication, a pharmaceutical, or a substance which isintended for use in diagnosis, cure, mitigation, treatment, orprevention of disease, or which is generally intended to affect thestructure or the function of the body of a mammal.

The invention also includes a method of preparing any of the variousformula (I) compounds of the invention. The method involves providing acombination of one or more of X¹ _(n), X², X³, X⁴ _(n), X⁵, or X¹X² in asolution, e.g., by dissolving the combination, e.g., one or more of X¹_(n, X) ², X³, X⁴ _(n), X⁵, or X¹X², in a solution; cooling thesolution; adjusting the pH of the solution to the neutral range, e.g., apH of 4-8, preferably pH 6-8, or between pH 7 and 8, inclusive; andpurifying the formula (I) compound from the solution. By “purifying” ismeant extracting, filtering, evaporating, precipitating, washing,recrystallizing, isolating by chromatography, or any other means ofisolating the desired Reversin compound from the reaction mixture. Themethod can further include an additional purification step to remove anyimpurities from the final product, e.g., a gel filtration step, or achromatographic step (see Methods, below). The method can also include,or further include, a step of active ester coupling, or a step ofdicyclohexylcarbodiimide condensation characterized by the followingparameters: cooling by ice-water, 10% excess of DCC, pH adjusted between7-8 with tertiary base (e.g. triethylamine, N-methylmolpholine,diisopropyl-ethylamine). In certain cases 1-hydroxybenzotriazoleadditive is used for activation and to avoid possible racemization (W.Lonig et al. Chem Ber. 103:2024, 1970, hereby incorporated byreference).

The invention also includes a method of reducing the activity of amultidrug transporter protein or its homologues in a mammal. The methodinvolves administering to the mammal an amount of any of the variousformula (I) compounds of the invention in a therapeutically effectiveamount. The method of reducing the activity of MDR1 can be used to lowerresistance to a drug, where the drug includes one or more, e.g., atleast one, two, or three drugs, which are chemotherapeutic drugs,antiparasitic drugs, or antibiotic drugs. By “mammal” is meant a human,a domesticated animal, e.g., a cat or a dog, or an agricultural animal,e.g., a cow, pig, sheep, horse, or poultry. A “chemotherapeutic drug”includes any drug intended to target and kill a tumor cell, e.g.,neoplastic, malignant, or benign tumor cell, in a mammal. An“antiparasitic drug” includes a drug intended to target the agent of aparasitic infection, e.g., ascaris, enterobium, hookworm, threadworm,tapeworm, schistosomes, whipworm, protozoa, e.g., intestinal orextraintestinal amebas, giardia, malaria, toxoplasma, or trichomonas. An“antibiotic drug” includes substances which inhibit or kill fungal orbacterial microorganisms, e.g., actinomycin. Examples of drugs withinthe scope of the invention include, but are not limited to, thesubstances listed in Table 1, as well as any chemotherapeutic,antiparasitic, and antibiotic drugs which clinically elicit, or whosetherapeutic effects are limited by, primary or secondary multidrugresistance caused by MDR1 (Ford et al. supra; hereby incorporated byreference).

Cytotoxic drugs which are extruded by the MDR1 protein or its homologuesinclude, but are not limited to, the compounds shown in Table 1.

TABLE I Drugs exported by the MDR1 protein Examples Anti-Cancer DrugsVinca alkaloids vinblastine, vincristine vindesine Anthracyclinesdoxorubicin, daunorubicin epirubicin Epipodophyllotoxins etoposideAntibiotics actinomycin D Others mitomycin C, taxol, topotecan,mithramycin Other cytotoxic agents Anti-microtubule drugs colchicine,podophyllotoxin Protein Synthesis inhibitors puromycin, emetine DNAintercalators ethidium bromide Toxic peptides valinomycin, gramicidinD,N-acetyl-leucyl leucyl-norleucinal (ALLN)

The method of reducing the activity of a MDR1 protein or its homologuesin a mammal is also used to facilitate administration of a drug throughmembranes which exclude various substances from a given type of cell ortissue. In particular, Reversins can be used to aid transport of a drugthrough the blood-brain barrier, or through the blood-testis barrier. By“blood-brain barrier” and “blood-testis barrier” is meant theendothelial lining of cells that are selectively permeable orimpermeable to substances circulating outside of the brain or testis,respectively.

A “multidrug transporter protein” (MDR1), as used herein, refers to aglycoprotein present on the membrane of many cell types which acts toextrude various substances from the cell. In humans, MDR1 is commonlyreferred to as the P-glycoprotein, P-170, or Pgp, and is encoded by themdr1 gene. Also included are homologues of MDR1 which are members of theMDR1 family of proteins in other organisms, e.g., prokaryotes or lowereukaryotes, e.g., bacterial, yeast, fungal, parasitic organisms, orother organisms that take up residence within a mammalian body.Homologues within the MDR1 family of proteins perform the same functionas MDR1 for cells of the other organisms, i.e., by transportinghydrophobic cytotoxic compounds out of the cell. Generally, the methodof the invention is intended to inhibit the activity of members of theMDR1 protein family which are shown by the assays below to be affectedby a Reversin compound.

A “therapeutically effective amount”, as used herein, refers to anamount that is effective at reducing the activity of the multidrugtransporter protein; an amount that is effective at lowering resistanceof the mammal to a drug or to a group of drugs; or an amount that iseffective for facilitating absorption of a drug through the blood brainbarrier. An “effective amount” can be calibrated by the assays below. By“facilitating” is meant enhancing the overall amount of the drug that isabsorbed, or the fraction of the drug that is absorbed.

As used herein, the term “reducing” means either partially or completelyinhibiting MDR1 activity. By “reducing” is also meant decreasing,lowering, or overcoming the effects of drug or multidrug resistance, sothat less drug is transported from the target cell, or so that a greaterconcentration of drug accumulates within the cell. As used herein, theterm “reducing” encompasses both treating and preventing the occurrenceof drug, e.g., multidrug, resistance in a mammal. The level of MDR1activity present in a cell or in a target tissue is measured by theassays provided below. This in turn permits accumulation of drug athigher concentrations in the cell than would be possible in the absenceof the Reversin compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

DETAILED DESCRIPTION

We first briefly describe the drawings.

Drawings

FIG. 1 is a schematic illustration of the MDR1 protein in its membraneenvironment.

FIG. 2 is a computer-generated illustration of the secondary structureof the Reversin 121 molecule, BOC-Asp(OBzl)-Lys(Z)-OtBu.

FIG. 3 is a graph showing drug-stimulation of human MDR1-ATPase activityin isolated insect cell membranes.

FIG. 4 is a graph showing stimulation of the vanadate-sensitive humanMDR1-ATPase activity in the isolated membranes of Sf9 cells by verapamiland Reversins.

FIG. 5 is a bar graph showing the effects of Reversins on Fura-2 uptakein NIH 3T3 fibroblasts.

FIG. 6 is a bar graph showing the inhibition of Fura-2 AM loading inMDR1-expressing fibroblasts by agents interacting with MDR1.

FIG. 7 is a photograph showing the effect of verapamil on dye loading inMDRl-expressing fibroblasts using single-cell imaging for fluorescentdye uptake. The upper panel shows control NIH 3T3 cells; middle andlower panels show MDR1-3T3 cells. In the lower panel the medium alsocontained 25 μM verapamil.

FIG. 8 is a graphic illustration of the fluorescent dye uptake ofcontrol cells (A) versus fluorescent dye uptake in the presence of 5 μMReversin 205 (B).

FIG. 9 is a bar graph showing the effect of various pretreatments andwashings on fluorescent dye uptake into 3T3-MDR cells.

FIGS. 10A and 10B are graphic illustrations of the effect of Reversin205 and verapamil on drug-sensitive and multidrug resistant culturedhuman tumor cells (K562) treated with adriamycin (A) or vincristine (B).K562 control, -+-K652+5 μMR, K562+10 μM Verap, K562 MDR, MDR+5 μMR, K562MDR+10 μMV.

FIGS. 11A and 11B are graphic illustrations of the effect of Reversins121 (A) and 205 (B) on the vinblastine-sensitivity of drug-sensitive(KB3) and multidrug-resistant (KBV1) cultured human tumor cells. KB3control, -+-KB3+2 ug/ml R, KB3 +10 ug/ml R, KBV1 control, KBV1+2 ug/mlR, KBV1+10 ug/ml R.

REVERSIN COMPOUNDS

The aim of the present invention is to provide chemical reversingagents, or chemosensitizers, to address the clinical problem ofmultidrug resistance. Toward this end applicants designed a set of novelhydrophobic peptide-based molecules called Reversins.

Reversins effectively inhibit expulsion of therapeutic drugs. They areeffective at low concentration, non-toxic, and reversible. Reversinsalso leave the organism without adverse side effects, and in some caseseven enhance the activity of the drug therapy itself. They can beprepared in high purity, in large quantities, and at relatively lowcost. Reversins are especially advantageous for preventing resistanceagainst anticancer drugs, without causing serious side-effects in othertissues, and without irreversibly disrupting the important naturalphysiological function of the MDR1 transport protein itself.

Methods of Preparation

Reversins are peptide derivatives consisting of naturally occurring Lamino acids with bulky aromatic or alkyl groups, and carboxamide orcarboxylic acid ester groups. The hydrophobic side-chains of themolecules enhance their interaction with the MDR1 transporter for whichthey are a substrate. The overall size of the molecule also influencesthis interaction. Dipeptides and tripeptides are preferred. Acomputer-analyzed secondary structure of a preferred dipeptide, Reversin121, is illustrated in FIG. 2.

In general, Reversins are synthesized by an appropriate fragmentcondensation reaction, depending on the chemical character of the aminoacid moiety in the compound being prepared. Reversins are synthesized bytraditional stepwise condensation methods (The Peptides: Methods ofpeptide synthesis, Eds. E. Schoder, K. Lubke, Acad. Press, NY, 1966; ThePeptides: Analysis, synthesis and biology, Ed. by E. Gross, J.Meienhofer, Acad. Press, NY 1979, each hereby incorporated byreference), or by automated solid-phase peptide synthetic methods, whichare relatively large-scale and inexpensive. Reversins are also preparedby methods known to those of ordinary skill in the art, and asexemplified by Examples 1-12, below.

Each of the Examples 1-12, below, are prepared from amino acids thatwere protected to a desired grade. The starting materials are dissolvedin apolar solvents, e.g., DMF, acetonitril, or DMSO. Condensationreactions are performed in solution, or by passage of the solution overa solid-phase column. The solution is cooled, for example to 0-10° C.,and the pH is adjusted to the neutral range with, e.g., triethylamine,N-methylmorpholine, trimethyalmine, or diisopropylethylamine, and theproduct is allowed to form slowly, e.g. by stirring overnight. Finally,the Reversin compound is purified from the reaction mixture by, e.g.,filtration of the precipitate, or by evaporating away the mother liquor,and the product is washed.

The final products can receive additional purification in order toremove any contaminants from the Reversin compound. The finalpurification step can include one or a combination of the followingprocedures: ethyl acetate; gel filtration; preparative high pressureliquid chromatography (HPLC); medium pressure column chromatography; orsilica gel column chromatography. The purity of the peptides obtained isdetermined by thin layer chromatography (TLC) analysis.

In the examples that follow, R_(f) values were obtained by TLC on Kieselgel sheets (DC Alufolien Merck) using the following solvent mixtures (inthe examples the solvent mixtures will be identified by the numberslisted below):

(1) acetone:toluene, 1:1

(2) chloroform:acetic acid:benzene, 85:10:5

(3) acetic acid:benzene, 1:7

(4) ethyl acetate:pyridine:acetic acid:water 240:20:6:11.

In general, Reversins are only slightly soluble in water (maximumsolubility is about 10 μg/ml), while freely soluble in dimethylsulfoxide (DMSO), glycerol, or ethanol. When testing a Reversin in an invitro cellular screening assay, the presence of serum in the culturemedia increases the solubility of the Reversin.

The following non-limiting examples are provided to illustrate methodsof preparing the Reversin compounds of the invention.

EXAMPLE 1 Preparation of BOC-Asp(OBzl)-Lys(Z)-OtBu (Reversin 121)

After dissolving 1 mmol of BOC-Asp(OBzl)-OH amino acid derivative (molewt.=323) in 50 ml of DMF, 1.1 mmoles of DCC (mole wt.=206) and then 1mmole of N6-carbobenzoxylysine tert.-butyl ester hydrochloride (molewt.=371) were added while stirring and cooling with ice water. The pHvalue of the reaction mixture was adjusted to 7-8 with triethylamine andthe mixture was stirred at room temperature overnight. The precipitatewas filtered off and the mother liquor was evaporated. After taking upthe residue in 150 ml of ethyl acetate, the extract was washed threetimes with 120 ml of 10% citric acid solution each, then three timeswith saturated sodium hydrogen carbonate solution, and finally threetimes with saturated saline solution. After drying the ethyl acetatephase over anhydrous sodium sulfate and filtering off the drying agent,the organic phase was evaporated and the residue was recrystallized from30% alcohol to give the title product. m.p.: 128-129° C., R_(f) (4)=0.9.The possible conformation of Reversin 121 is shown in FIG. 2.

The molecular mass of Reversin 121 is 641.5.

EXAMPLE 2 Preparation of succinyl-bis[Glu(OBzl)-OBzl]

To a solution containing 1 mmole of dibenzyl glutamate hydrochloride(mole wt.=362) in 50 ml of DMF, 0.5 mmole of bis(pentafluorophenyl)succinate was added while stirring and cooling with ice water. The pHvalue of the reaction mixture was adjusted to between 7 and 8 withtriethylamine, and the mixture was stirred at room temperatureovernight. Then the precipitate was filtered off, the mother liquor wasevaporated and the residue was taken up in 150 ml of ethyl acetate. Theorganic solution was successively washed three times with 120 ml of 10%citric acid solution each, three times with saturated sodium hydrogencarbonate solution, and finally with saturated saline solution. Afterdrying the ethyl acetate the phase over anhydrous sodium sulfate andfiltering off the drying agent, the organic phase was evaporated, andthe evaporation reside was recrystallized from aqueous alcohol to givethe white crystalline title product, m.p.: 106-107° C., R_(f) (4)=0.9.

EXAMPLE 3 Preparation of Z-D-Glu(OBzl)-D-Asp(OBzl)-OBzl

To a solution containing 1 mmol of the amino acid derivativeZ-D-Glu(OBzl)-OH (mole wt.−371) in 50 ml of DMF, 1.1 mmoles of DCC andthen 1 mmol of H-D-Asp(OBzl)-OBzl hydrochloride (mole wt.−349) wereadded while stirring and cooling with ice water. The pH value of thereaction mixture was adjusted to between 7 and 8 with triethylamine, andthe mixture was stirred at room temperature overnight. After filteringoff the precipitate, the mother liquor was evaporated, and the residuewas taken up in 150 ml of ethyl acetate. The organic solution wassuccessively washed three times with 120 ml of 10% citric acid solutioneach, three times with saturated sodium hydrogen carbonate solution, andfinally with saturated saline solution. After drying the ethyl acetatephase over anhydrous sodium sulfate, the drying agent was filtered offand the organic phase was evaporated to give the title compound as aslightly yellow crystalline product. R_(f) (4)=0.9.

EXAMPLE 4 Preparation of DBTA-bis(D-Phe-Trp-OMe)

To a solution containing 1 mmole of the dipeptide hydrochlorideD-Phe-Trp-OMe (mole wt.=401.8) in 50 ml of DMF, first 1.1 mmoles of DCC,and then subsequently 0.5 mmole of DBTA-(OPFP)₂ active ester (molewt.=696), were added while stirring and cooling with ice-water. The pHvalue of the reaction mixture was adjusted to between 7 and 8 withtriethylamine and the mixture was stirred at room temperature overnight.Then, the precipitate was filtered off, the mother liquor wasevaporated, and the residue was taken up in 150 ml of ethyl acetate. Theorganic solution was successively washed three times with 120 ml of 10%citric acid solution each, 3 times with saturated sodium hydrogencarbonate solution, and finally three times with saturated salinesolution. After drying the ethyl acetate phase over anhydrous sodiumsulfate and filtering off the drying agent, the organic phase wasevaporated to obtain the title compound as a foam-like amorphousevaporation residue. m.p.: 79-80° C., R_(f) (4)=0.9.

EXAMPLE 5 Preparation of DBTA-[Glu(OBzl)₂]₂

To a solution containing 1 mmole of the active ester DBTA-(OPFP)₂ (molewt.=696) in 50 ml of DMF, 2 mmoles of Glu(OBzl)₂ tosylate (mole wt.−500)and then 0.28 ml (2 mmoles) triethylamine, were added while stirring andcooling with ice water. The pH value of the reaction mixture wasadjusted to between 7 and 8 with triethylamine and the mixture wasstirred at room temperature overnight. Then, the precipitate wasfiltered off and after evaporating the mother liquor the residue wastaken up in 150 ml of ethyl acetate. The organic solution wassuccessively washed three times with 120 ml of 10% citric acid solutioneach, three times with saturated sodium hydrogen carbonate solution, andfinally three times with saturated saline solution. After drying theethyl acetate phase over anhydrous sodium sulfate, the drying agent wasfiltered off and the organic phase was evaporated to give the titlecompound as an oily residue. R_(f) (4)=0.9.

EXAMPLE 6 Preparation of N^(α), N^(ε)-bis[BOC-Glu(OBzl)]-Lys-OMe(Reversin 205)

Method A: To a solution containing 1 mmol of the amino acid derivativeBOC-Glu(OBzl)-OH (mole wt.=337) in 50 ml of DMF, 1.1 mmoles of DCC and 1mmoles of the amino acid derivative Lys-OMe dihydrochloride (molewt.=228) were added while stirring and cooling in ice water. Afteradjusting the pH value to between 7 and 8 with triethylamine, thereaction mixture was stirred at room temperature overnight. Then, theprecipitate was filtered off and the mother liquor was evaporated. Theresidue was taken up in 150 ml of ethyl acetate and successively washedthree times with 120 ml of 10% citric acid each, three times withsaturated sodium hydrogen carbonate solution, and finally three timeswith saturated saline solution. After drying the ethyl acetate phaseover anhydrous sodium sulfate, the drying agent was filtered off and theorganic phase was evaporated. After recrystallizing the evaporationresidue from the aqueous alcohol, the title compound was obtained as aslightly yellow crystalline product. m.p.: 79-81° C., R_(f) (1)=0.7,R_(f) (4)=0.95.

Method B: A second method of preparing N^(α),N^(ε)-bis[BOC-Glu(OBzl)]-Lys-OMe is according to the procedure ofExample 11, except that 5.03 g of the amino acid active esterBOC-Glu(OBzl)-OPFP was used as the starting material, and the otherconstituents were used in the amounts of 1.15 g of Lys-OMedihydrochloride, 20 ml of DMF and 1.38 ml of triethylamine.

The molecular mass of Reversin 205 is 875.5.

EXAMPLE 7 Preparation of BOC-Tyr(Bzl)-Tyr(Bzl)-OMe

To a solution containing 986 mg of the amino acid derivativeBOC-Tyr(Bzl)-ONp (mole wt.=491) in 50 ml DMF, first 682 mg ofH-Tyr(Bzl)-OMe. OMe hydrochloride salt (mole wt=384), and then 0.28 mltriethylamine were added while stirring and cooling with ice water. ThepH value of the reaction mixture was adjusted to between 7 and 8 withtriethylamine, and the mixture was stirred at room temperature for 24hours. After evaporating the mother liquor the residue was taken up in50 ml of ethyl acetate. The organic solution was successively washedthree times with 2N KHSO₄ solution, three times with saturated sodiumhydrogen carbonate solution, and three times with saturated salinesolution. After drying the ethyl acetate phase over anhydrous sodiumsulfate, the drying agent was filtered off. The organic phase wasevaporated to give an oil which can be crystallized from the mixture ofethanol:water (7:3) to obtain the title compound. R_(f)(1):0.95;(R_(f)(4):0.9; m.p.=161-163° C.

EXAMPLE 8 Preparation of BOC-D-Ser(Bzl)-Lys(Z)-OtBu

To a solution containing 10 mmoles of the amino acid derivativeBOC-D-Ser(Bzl)-OH (mole wt.=294) in 20 ml DMF, were first added 11mmoles of DCC (mole wt.=206), 3.5 g of H-Lys(Z)-OtBu hydrochloride salt(mole wt.=336). Then 1.13 ml of triethylamine were added while stirringand cooling in ice water. The pH value of the reaction mixture wasadjusted to between 7 and 8 by using triethylamine, and the mixture wasstirred at room temperature for 24 hours. After evaporating the motherliquor the residue was taken up in 50 ml of ethyl acetate. The organicsolution was successively washed three times with 2N KHSO₄ solution,three times with saturated sodium hydrogen carbonate solution, andfinally three times with saturated saline solution. After drying theethyl acetate phase over anhydrous sodium sulfate, and filtering off thedrying agent the organic phase was evaporated to obtain the titlecompound as an oil which is triturated in petroleum ether.R_(f)(4):0.85; R_(f)(5): 0.65.

EXAMPLE 9 Preparation of BOC-Glu(OBzl)-Lys(Z)-OtBu

To a solution containing 2.12 g of BOC-Glu(OBzl)-OFFP amino acid activeester (mole wt.=503) in 20 ml DMF, first 744 mg H-Lys(Z)-OtBuhydrochloride salt (mole wt.=371), then 0.28 ml triethylamine were addedwhile stirring and cooling by ice-water. The pH value of the reactionmixture is adjusted between 7 and 8 by using triethylamine and themixture is stirred at room temperature for 24 hours. After evaporatingthe mother liquor the residue is taken up in 30 ml of ethyl acetate. Theorganic solution is successively washed three times with 10% citricacid, three times with saturated sodium hydrogen carbonate solution, andfinally three times with saturated saline solution. After drying theorganic phase over anhydrous sodium sulfate, the drying agent wasfiltered off and the organic phase was evaporated to give an oil whichwas then triturated in petroleum ether and crystallized from ethanolwith water to obtain the title compound. R_(f)(1):0.95; m.p.=79-81° C.

EXAMPLE 10 Preparation of BOC-Glu(OBzl)-Lys(2)-OMe

To a solution containing 2.1 g of the amino acid active esterBOC-Glu(OBzl)-OPFP (mole wt.=503) in 10 ml DMF, was first added 1.3 g ofH-Lys(Z)-OMe hydrochloride salt (mole wt.=329). Then 0.28 ml oftriethylamine were added while stirring and cooling with ice water. ThepH value of the reaction mixture was adjusted to between 7 and 8 withtriethylamine, and the mixture was stirred at room temperature for 24hours. After evaporating the mother liquor the residue was taken up in50 ml of ether. The organic solution was successively washed three timeswith 2N KHSO₄ solution, three times with saturated sodium hydrogencarbonate solution, and finally three times with saturated salinesolution. After drying the organic phase over anhydrous sodium sulfate,the arying agent was filtered off, and the organic phase was evaporatedto give an oil which was triturated in petroleum ether and crystallizedfrom ethanol with water to obtain the title compound. R_(f)(1)=0.80;R_(f)(4):0.85; R_(f)(5):0.90; m.p.=102-105° C.

EXAMPLE 11 Preparation of N^(α),N^(ε)-bis[BOC-Lys (BOC)]-Lys-OMe

To a solution containing 5.12 g of the amino acid active esterBOC-Lys(BOC)-OPFP (mole wt.=512) in 50 ml DMF, was first added 1.15 g ofLys-OMe dihydrochloride salt (mole wt.=231). Then 1.38 ml oftriethylamine were added while stirring and cooling with ice water. ThepH value of the reaction mixture was adjusted to between 7 and 8 withtriethylamine, and the mixture was stirred at room temperature for 24hours. After evaporating the mother liquor the residue was taken up in50 ml of ether. The organic solution was successively washed three timeswith 2N KHSO₄ solution, three times with saturated sodium hydrogencarbonate solution, and finally three times with saturated salinesolution. After drying the organic phase over anhydrous sodium sulfate,the drying agent was filtered off, and the organic phase was evaporatedto give the title compound as an oil. R_(f)(1):0.90; R_(f)(4):0.90.

EXAMPLE 12 Preparation of N^(α),N^(ε)-bis[Z-Glu(OtBu)]-Lys-OMe

N^(α),N^(ε)-bis[(Z-Glu(OtBu)]-Lys-OMe was prepared according to theprocedure of example 11, except that 2.5 g of the amino acid activeester Z-Glu(OtBu)-OPFP was used as the starting material, and otherconstituents were used in the amounts of 0.6 g of Lys-OMedihydrochloride, 25 ml DMF, and 0.1 ml triethylamine. The title compoundis a white crystalline material. R_(f)(1):0.70; R_(f)(4):0.75;m.p.=83-84° C.

Methods for Demonstrating the Efficacy of Reversins

Any of the various Reversin compounds of the invention can be screenedfor their ability to reduce the activity of the MDR1 protein accordingto the following in vitro and in vivo methods. In addition to theinstructions and experiments provided below, each method is supported byone or more publications, each of which is hereby incorporated byreference.

A. In vitro Methods

Two test systems were developed to specifically assess the ability of aReversin compound to interact with the human MDRl protein. The firstsystem measures the ATPase activity of MDR1, while the second systemmeasures the level of a fluorescent indicator extruded by the MDR1protein.

1. ATPase Assay

In the first assay, MDR1-ATPase activation reflects the interaction andrelative affinity MDR1 has for a candidate compound. The MDR1-ATPase isstimulated by cytotoxic drugs, e.g., vincristine, while it isinsensitive to chemicals that are not transported by the MDRl protein.The MDR1-ATPase is also stimulated by known chemosensitizing agents,such as the multidrug-resistance reversing agents verapamil and quinine(FIG. 3), probably competing with drug extrusion of the multidrugtransporter. Thus, by measuring this MDR1-ATPase a relatively simple invitro assay system became available for assessing direct druginteractions with the MDR1 protein (Sarkadi et al. J. Biol. Chem.267:4854-4858, 1992).

The assay was developed by expressing the human MDR1 protein inSpodoptera frugiperda insect cells. The cultured cells were infectedwith a baculovirus into which the cDNA of human MDR1 was geneticallyengineered. The recombinant virus-infected cells produce a large amountof the MDR1 protein, properly folded and functionally inserted into themembrane. Additional details regarding construction of these recombinantstrains are provided by Germann et al. (Biochemistry 29:2295-2303, 1988)and Sarkadi et al. (1992 supra).

Measurements of the effect of Reversins on the MDR1 ATPase activity areperformed as follows, and according to the methods provided by Sarkadiet al. (1992, supra).

MDR1 ATPase measurements:

Spodoptera frugiporda (Sf9) cells were infected with a recombinantbaculovirus carrying the human MDR1 gene, and cultured according to theprocedures described previously (Germann et al. 1990 supra; Sarkadi etal. 1992 supra). The virus-infected Sf9 cells were harvested, and theirmembranes isolated and stored as described (Sarkadi et al. J. Biol.Chem. 267:4854-4858, 1992). The amount of ATP consumption measured inthese membranes reflects the ATP-dependent transport function of themultidrug transporter. ATPase activity of the isolated Sf9 cellmembranes was estimated by measuring inorganic phosphate (P_(i))liberation. To do this, a membrane suspension (about 10 μg of membraneprotein) was incubated at 37° C. in 0.1 ml of a medium containing 50 mMTris-Mes (pH 6.8), 2 mM EGTA, 2 mM DTT, 50 mM KC1, and 5 mM Na-azide.The ATPase reaction was started by the addition of 5 mM MgATP. Thereaction was stopped by the addition of 0.1 ml of 5% SDS solution, andthe amount of P_(i) determined immediately. ATPase activity wasestimated by the difference obtained in P_(i) levels by a sensitivecalorimetric assay between zero minutes (reaction stopped immediatelywith SDS) and 20 minute incubation periods. The data points show themeans of triplicate determinations in a representative experiment. Thedifferences between the ATPase activities measured in the absence andpresence of vanadate (100 μM) are plotted. Isolated membranes ofuninfected or B galactosidase infected Sf9 cells had no drug-stimulatedATPase activity (FIG. 3).

When Reversins 121 and 205 were tested in the ATPase assay system, theygreatly stimulated the MDR1-ATPase, but were effective at significantly(one to two orders of magnitude) smaller concentrations than the knownreversing agents verapamil and quinine. The half-maximal activatingconcentration (K_(a)) of verapamil was approximately 1 μM, while theK_(a) value for Reversin 121 was approximately 60 nM and for Reversin205 this value 20 was about 30 nM (FIG. 4). Thus, the multidrugtransporter seems to interact with Reversins with an exceptionally highaffinity. As shown in FIG. 4, a strong inhibition of the MDR1-ATPase wasobserved at higher concentrations (above 1 μM) of Reversin 205.

2. Fluorescence Assay:

The second assay is based on the measurement of fluorescent dye uptakeinto intact cells. Fluorescent dyes are often used to indicateintracellular calcium or pH changes. An effective technique for cellulardye loading is the application of acetoxy-methylester (AM) derivatives.These hydrophobic dye esters are non-fluorescent outside the cell arecleaved by intracellular esterases into hydrophilic fluorescent freeacids. This intracellular “trapping” of the free dye and the continuousinward gradient of the AM compounds results in the accumulation of largeamounts of fluorescent indicator inside the cell.

Cell culturing:

NIH 3T3 cells were cultured under standard conditions in D-MEM medium.MDR1-transfected cells (NIH MDR1 G185) were prepared and characterizedfor their drug-resistant properties as described (Ambdukar et al. Proc.Natl. Acad. Sci. USA 89:8472-8476, 1992; Bruggemann et al. J. Biol.Chem. 267:21020-21026, 1992; Sarkadi et al. 1992 supra). Before eachexperiment the cells were trypsinized, then washed and stored in D-MEMmedium at 37° C. KBV1 (MDR1⁺) and KB3 (MDR1⁻) human tumor cells werealso cultured in D-MEM, which K562 human tumor cells were grown in RPMImedium, supplemented with 10% FCS.

The effects of Reversins 121 and 205 on MDR1 function in intact cellswas examined by a fluorescent dye extrusion assay (FIG. 5). Mouse NIH3T3 fibroblasts, transfected with human MDR1 cDNA and expressing humanMDR1 protein, actively extrude the hydrophobic AM derivatives offluorescent dyes, e.g., Fura2-AM (Homolya et al. J. Biol. Chem.29:21493-21496, 1993; Hollo et al. Bioch. Biophy. Acta. 1191:384-388,1994). Similar experiments can be performed with MDR1-expressing humantumor cells. Verapamil, vincristine, and Reversins 121 and 205 inhibitthis dye extrusion, most probably by competing with the dye on thetransporter. In the experiments shown here maximally effectiveconcentrations of Reversins were used. However, Reversins also act in atleast one order of magnitude smaller concentrations than verapamil.

Human MDR1-transfected mouse fibroblasts and MDR1-expressing human tumorcells actively extrude the AM forms of several fluorescent indicators,lowering the level of intracellular fluorescence in cells with activemultidrug transport. Thus the accumulation in such cells of fluorescentdye is strongly inhibited. This MDR1-specific dye-AM extrusion isblocked by competing substrates and inhibitors of the MDR1 transporter,e.g., by verapamil, vincristine, sodium orthovanadate, and a monoclonalanti-MDR1 antibody. In contrast, these agents have no effect on dyeaccumulation in fibroblasts which do not overexpress MDR1 (FIG. 6). Seealso Kessel et al. (Cancer Res. 51:665-670, 1991); Neyfakh et al. (Exp.Cell. Res. 174:168-176, 1988); and Sarkadi et al. (J. Biol. Chem.268:21493-21496, 1993).

Fluorescence studies:

Dye uptake was measured by incubating 2×10⁶ cells/ml D-MEM medium at 37°C. in the presence of 0.5 μM Fura-2 AM (added in 5 mM stock solution inDMSO), then rapidly spinning the cells (15 sec, 12,000×g), and rinsingthe pellet with HPM1 medium (containing 120 mM NaCl, 5 mM kcl, 0.4 mMMgCl₂, 0.04 mM CaCl₂, 10 mM HEPES-Na (pH 7.4), 10 mM NaHCO₃, 10 mMglucose, and 5 mM Na₂HPO₄). The cells were resuspended in 2 ml HPM1 andfluorescence was measured with rapid stirring in a Hitachi F-4000fluorescent spectrophotometer. The excitation wavelength was 340 nm, andemission was measured at 410 nm. Maximum fluorescence and dyeconcentration were measured after the addition of 0.5% Triton X-100 and2 mM CaCl₂ to the medium. The dye concentration was calibrated based onthe measurements of free acid dye fluorescence in the same instrumentunder identical conditions.

Flow-cytometric measurements:

Fluorescence measurements were done in 60 sec scanning periods using aCytoronabsolut instrument (Ortho Diagnostic Systems, NJ). The excitationwas set to 488 nm. Green fluorescence was measured with a filter with arange of 515-548 nm, while red fluorescence was measured above 620 nm.

With this method drug-interactions with the MDR1 protein can be measuredby following cellular fluorescence allowing a flow cytometric or singlecell imaging detection of the function of MDR1 in tumor cells. As shownin the single cell images of FIG. 7, fibroblasts expressing the MDR1protein, in contrast to the control cells, are not loaded with afluorescent dye, while verapamil, which inhibits the multidrugtransporter, restores dye uptake.

When assaying the effect of Reversin molecules on the fluorescent dyeuptake and the drug-resistance in various MDR1-transfected andmultidrug-resistant tumor cell lines in vitro, these experimentsindicated a strong inhibitory action on drug extrusion by lowconcentrations of Reversins, again showing a specific interaction of themultidrug transporter with these molecules.

Binding Experiments:

The relative ability of Reversins to bind to the MDR1 protein, or to beremoved from the cell membrane, is measured by “wash-out experiments”(FIG. 9). NIH 3T3-MDR1 cells were pre-treated with 15 μM verapamil, 5 μMReversin 121, or 5 μM Reversin 205 for 5 minutes, which produced acomplete inhibition of dye extrusion by MDR1 with 1% serum in the media.The cells were washed once with the standard incubation media and a 5min centrifugation at 800×g. Dye uptake was then measured with orwithout the addition of 15 μM verapamil during the uptake period. Asshown, preincubation with verapamil had no effect on dye uptake, whileboth Reversin 121 and Reversin 205 had a major effect even after washingthe cells. Thus verapamil could be eliminated by a single wash, while121 and 205 remained effectively bound to MDR1.

Assessing Reversin Activity by its Ability to Enhance the CytotoxicityCaused by Other Agents

Another method for assessing the ability of Reversins to inhibit drugresistance is to measure the cytotoxicity of drugs inmultidrug-resistant human tumor cell lines. Since MDR1 normally lowersthe concentration of cytotoxic agents to subtoxic concentrations byextruding them from the cell, inhibition of MDR1 would be expected toenhance cytotoxicity.

In these studies, adriamycin, vincristine, and vinblastine, inoriginally ineffective concentrations, become effectively cytotoxic inthe presence of 1-10 μg/ml of Reversins 121 or 205. FIGS. 10A and 10Bpresent such an experiment for Reversin 205 using K562 humanerythroblastoid tumor cells and their adriamycin-selectedmultidrug-resistant subline. FIGS. 11A and 11B are similar experimentswith the intestinal tumor cells KB3 and KBV1, the latter being amultidrug resistant subline. In the experiment shown in FIG. 11(A),Reversin 121 was found to be cytotoxic in the MDR1-expressing cells (butnot in the control cells) even without the addition vinblastine. Such acollateral toxic effect (which may be due to the ATP-consuming futilefunctioning of the drug transporter) can be greatly advantageous intreating drug-resistant tumors.

Application of the in Vitro Fluorescent Assay for Clinical Diagnosis

By using the some fluorescent dye extrusion assay described above the invitro effects of Reversins on the multidrug-resistant leukocytes of aleukemic patient were studied. Cells were isolated by withdrawing bloodfrom the patient, and isolating white blood cells by centrifugation.Cells can be isolated from other types of tumors by biopsy. A flowcytometer was used to measure fluorescent dye loading of individualcells, which had been previously shown to express MDR1, in the absenceor presence of Reversin 205 (FIG. 8). The uptake of fluorescent dyeunder these conditions models the uptake of cytotoxic drugs into thetumor cells. Fluorescent dye uptake was measured at 37° C. for 10minutes. In FIG. 8, the MDR1 containing cells in the control experimenthad a low fluorescent dye uptake (A), while the addition of 5 μMReversin 205 blocked dye extrusion by MDR1 (B). Reversin 205 therebysignificantly increased dye uptake and yielded a uniformly highfluorescence in the leukocytes. Further details for the method areprovided by Hollo et al., supra.

Addition in vitro methods:

Additional in vitro methods of screening the ability of a Reversincompound to act as a chemosensitizing agent for the reversal ofmultidrug resistance are provided by Ford et al. supra, at Tables 1-6.

B. In vivo Methods

In vivo animal studies for the effectiveness of Reversins can beconducted using any suitable animal model system known to those skilledin the art. One example of an appropriate system is the mouse P388leukemia model system (Tsurouo et al., supra). This animal model iswidely accepted for testing the effect of cytotoxic antileukemic agentsor response-modifier compounds.

Inbred (DBA×black F1) mice received 10⁶ P388 leukemia cells byintraperitoneal injection. The survival of the mice was followed. Themice were injected with control P388 cells, as well as with P388 cellsthat had been selected under drug exposure. Drug exposure inducedover-expression of the MDR1 protein (P388-MDR cells).

In one trial, the mice had a mean survival time of 14-16 days (they diein a generalized leukemia caused by the P388 cells). The mice injectedwith the P388 cells were treated with doxorobicin (adriamycin) in a doseof 1 mg/kg/day for 6 days after the injection of the P388 cells. Theresults showed that adriamycin prolonged the survival time in the caseof mice injected with control P388 cells, exceeding the period of 30days. In contrast, adriamycin therapy did not significantly prolong thelifetime of mice injected with P388-MDR cells. Reversin can beadministered to the animals in a dose of 2 mg/kg, a level which has notoxic effect on the control animals, as discussed above.

Another test system is to conduct similar trials using human xenograftsin nude mice. Human erythroleukemia (K562) cells are injected intotolerant mice. The developing leukemia is treated with cytotoxiccompounds with or without a Reversin candidate compound.

Another animal model system for testing the ability of a Reversincompound to reverse multidrug resistance is to use a transgenic mousewhich expresses the human MDR1 gene, e.g., in its bone marrow (Pastan etal. FASEB Jour. 5:2523-2528, 1991).

Human clinical trials can be performed according to methods known tothose skilled in the art. For example, Dalton et al. provide methods oftesting the chemosensitizer verapamil for its ability to modifyresistance to a chemotherapeutic (Jour. Clin. Oncol. 7:415-424, 1989).Additional methods for conducting human clinical trials of Reversinsinclude, but are not limited to, those provided by Berenbaum et al.(Pharmacol. Rev. 41:93-141, 1989); Benson et al. (Cancer Treat. Rep.69:795-799, 1985); Cairo et al. (Cancer Res. 49:1063-1066, 1989); Fineet al. (J. Clin. Oncol. 5:489-10 495, 1987); Frishman et al. (J.Cardiol. 50:1180-1184, 1982); Miller et al. (J. Clin. Oncol. 6:880-888,1988); Ozols et al. (J. Clin. Oncol. 5:641-647, 1987); and Presant etal. (Am. J. Clin. Oncol. 9:355-357,1986).

Therapeutic Use of Reversins

The Reversins of the invention can be used therapeutically to inhibitthe in vivo activity of the MDR1 protein in a patient experiencing drugresistance or poor drug absorption. An effective amount of the Reversincan be administered intravenously to the patient, or administered orallyaccording to conventional methods, in the form of a capsule, liquid,tablet, powder, or pill. Reversin can also be incorporated into animplanted or orally administered slow release device.

Reversin can be prepared for therapeutic use by mixing the compound withpharmaceutical carriers and/or additives that aid solubility,absorption, flavor, or texture to the vehicle or its contents, e.g.,physiological saline, oil, e.g., refined soy bean oil, gelatin,glycerin, or purified water.

An appropriate dosage is between 50 μg/kg and 100 mg/kg. An effectiveand safe dosage can be determined by conventional methods or by themethods taught herein, or by calibration to a given patient on anindividual basis.

It has been found that compounds of the formula (I) according to ourinvention are capable of stimulating, in a concentration of 0.03 μM, orof inhibiting, respectively, in concentrations of 1 to 5 μM, theactivity of the MDR1 protein. The stimulating concentrations are by 1 to3 orders, the inhibitory concentrations are by 1 to 2 orders lower thanthe corresponding concentrations of substances known from theliterature, e.g., verapamil.

The safety of Reversins for human administration can be confirmed inappropriate animal models. For example, the in vivo acute and subacutetoxicity of Reversins was examined in laboratory rats. Reversins 121 and205 solutions were prepared and administered as follows. Type Asolutions contained 50 μg/ml Reversin 121 or 205 in 20% ethanol inphysiological saline. Type B solutions contained 1 mg/ml in glycerol,containing 10% ethanol. Type A solutions (0.5 ml) were administeredintravenously to three laboratory rats weighing 200-250 g each (about100-125 μg/kg) twice daily for 3 days. Type B solutions (0.5 ml) weregiven to similar rats (2-2.5 mg/kg) by intraperitoneal injection, twicedaily for three days. Control rats received the same solutions withoutthe Reversins. During the three days of injections and in a two-weekfollow-up period, no acute or subacute toxicity of the compounds wasobserved.

Additional toxicity testing can be performed by the methods of Pastan etal. (Proc. Natl. Acad. Sci. USA 85:4486-4490, 1988) and Tsuruo et al.(Cancer Res. 43:2905-2910, 1983).

Other embodiments are within the claims set forth below.

What is claimed is:
 1. A method for reducing the activity of themultidrug transporter protein in a mammal, said method comprisingadministering to said mammal an effective amount of a compoundBOC-Asp(OBzl)-Lys(Z)-OtBu, or N^(α),N^(ε)-bis[BOC-Glu(OBzl)]-Lys-OMe. 2.The method of claim 1, wherein said method is used to lower resistanceto a drug.
 3. The method of claim 2, wherein said drug is selected fromthe group consisting of a chemotherapeutic drug, an antiparasitic drugand an antibiotic drug.
 4. The method of claim 1, wherein said method isused to facilitate administration of a drug through the blood-brainbarrier or through the blood-testis barrier.
 5. The method of claim 1,wherein said compound of the formula (I) is co-administered with a drug.6. The method of claim 5, wherein said drug is selected from the groupconsisting of a chemotherapeutic drug, an antiparasitic drug and anantibiotic drug.
 7. A peptide compound selected from the groupconsisting of BOC-Asp(OBzl)-Lys(Z)-OtBu, succinyl-bis[Glu(OBzl)-OBzl],Z-D-Glu-(OBzl)-D-Asp(OBzl)-OBzl, DBTA-bis(D-Phe-Trp-OMe),DBTA-[Glu(O-Bzl)₂], N^(α),N^(ε)-bis[BOC-Glu(OBzl)]-Lys-OMe,BOC-Tyr(Bzl)-Tyr-(Bzl)-OMe, BOC-D-Ser(Bzl)-Lys(Z)-OtBu,BOC-Glu(OBzl)-Lys(z)-OtBu, N^(α),N^(ε)-bis[BOC-Lys(BOC)]Lys-OMe andN^(α),N^(ε)-bis[Z-Glu(OtBu)]Lys-OMe.
 8. A composition comprising thecompound defined in claim 7 which is BOC-Asp(OBzl)-Lys(Z)-OtBu orN^(α),N^(ε)-bis[BOC-Glu-(OBzl)]-Lys-OMe and a pharmaceuticallyacceptable carrier.
 9. A composition comprising the compound defined inclaim 7 which is BOC-Asp(OBzl)-Lys(Z)-OtBu orN^(α),N^(ε)-bis[BOC-Glu-(OBzl)]-Lys-OMe and a drug.